There are a number of diverse methods that have been suggested and implemented for the release of drugs over a sustained period of time in vivo. Many of these involve the physical entrapment of the drug by the fashioning of unique dosage forms that include enteric coatings, microencapsulation and so forth.
Another example utilizes polyethyleneglycol (PEG) irreversibly covalently linked to a polypeptide. Potential drawbacks with this system are that it may be immunogenic, and the PEG units may mask the polypeptide, effectively removing it from recognition by its receptor or otherwise interfering with its normal in vivo expression of biological activity.
Other methods involve the use of polysaccarides as drug carriers, reaction of bioactive agents with preformed polymers, and the use generally of carriers to carry the drug entity and deliver it variously over sustained periods in vivo. These methods generally involve irreversible chemical modifications of the polypeptide with predictable, and perhaps undesirable biological consequences. Attention in this regard is directed to Drug Delivery System, edited by Juliano, Oxford University Press, New York (1980); Medical Applications of Controlled Release, Vol. 1, editors Langer and Wise, CRC Press, Inc., Boca Raton, Fla. (1984), and Controlled Release Technology, Pharmaceutical Applications, editors Lee and Good, American Chemical Society, Washington, D.C. (1987).
Bernstein, et al., in J. National Cancer Institute 60, 379 (1978) coupled daunomycin with dextrans of various molecular sizes. In each instance, the unstable link of the antibiotic with the aldehyde of the oxidized dextran was immediately stabilized by reduction with sodium borohydride.
The foregoing dextran coupling is reported in the aforementioned Drug Delivery Systems text where there are references to several researchers in the area, using dextrans as carriers and more particularly periodate oxidation as the conjugation method (see particularly pages 261, 262 and 290 et seq.).
Odya, et al., one group of the cited researchers, [Biochemical Pharmacology 27, 173 (1978)], refers to the couplings of a number of polypeptides with oxidized dextran. In each case, subsequent treatment with sodium borohydride served to reduce both the reactive aldehyde groups remaining on the dextran and the unstable adduct formed when the proteins reacted with the aldehyde groups, resulting from the oxidized dextran, to produce stable secondary amines.
The reaction described in the foregoing references is known and is referred to commonly as reductive alkylation as a convenient method for converting amino groups in polypeptides to their alkylamine derivatives through the reduction of the adduct that forms between the amino groups and aldehydes as shown by the following reaction: ##STR1## This reaction is also described, for example, by Jentoft et el., Methods in Enzymology 91, 570 (1983) and in U.S. Pat. No. 3947352 (see particularly FIG. 2).
U.S. Pat. No. 4745180 and patent application WO 90/01332 describe polypeptide-heparin conjugates wherein the polypeptide is described as being covalently bonded via one or more lysine residues to heparin, and more particularly through the free epsilon amino group of the lysine group. This conjugation is brought about by the reactive aldehyde group of the heparin fragment forming the labile adducts that, as referred to above, are converted into stable secondary amines under reducing conditions.
These latter applications demonstrate the collective convention that because the intermediate imine conjugates (adducts) were inherently unstable--being labile under neutral aqueous solutions not unlike physiological conditions--they were to be avoided. Hence, they were eliminated by reduction (hydrogenation).